三维血管瘤血管生成体外模型的建立

A three-dimension In Vitro Human Model of Hemangioma and initial application

目的建立三维血管瘤血管生成体外培养模型。方法将10例新鲜的增殖期血管瘤组织块置于纤维蛋白凝胶中以双层夹心法建立三维血管瘤血管生成体外培养模型。于血管新生后3d加入平阳霉素（PYM，1×10^2mg／L）做干预实验。镜下观察新生血管情况，sP免疫组织化学法检测第Ⅷ因子相关抗原（FⅧR—Ag）、透射电镜观察内皮细胞超微结构鉴定新生血管。结果血管瘤组织培养4～7d后芽生出细小血管，至第8～10天长成枝丫状血管样结构，第11～14天之后血管样结构生长渐缓停滞萎缩。PYM作用1～2d后血管样结构很快萎缩。组织周边新生树枝状结构经鉴定为血管内皮细胞。结论该模型部分程度上可代表血管生成、萎缩的过程，但细小血管样结构的芽生时间不等，萎缩较快，与体内血管瘤血管仍有一定区别。

Objective To create a three dimension（3D） in vitro model for angiogenesis of hemangioma and to make initial use of the model. Methods A small fragment of hemangioma biopsy is embedded in fibrin gel in a well of culture plates and incubated in a serumree, buffered-salt, minimal medium to set up the three-dimesnsion（3D） in vitro model for angiogenesis of hemangioma. The model is cultured in the culture box of 5% C02,37 ℃ and saturated humidity. And pingyangmycin as an interference factor is used to observe its effect of blood vessels. We observed the model by photics microscope and fluorescence microscope and HE staining. Results In the model,a complex network of microvessels grows out from the 3 tissue fragments from the day 4th to 7th day after culture,and on the 8th to 10th day after culture a compact network of microvessels come into being and microvessels furcating and crossoverring, then tending to be stationary. Microvessels grow slowly to stagnate on the llth to ldth day after culture. The compact network around the tissue fragment was confirmed to be blood vessels by immunohistochemistry and electron microscopy. Microvessels grow fastly to stagnate after 1 day to 2 day putting pingyangmycin into the model. The expression of the factor Ⅷ associated antigen was positive in the microvessels analyzed by SP immunohistochemistry. The cells in the microvessels were the vascular endothelial cells observed by transmission electron. Conclusion This model represented the in vivo status of hemangioma partly and could be an acceptable model for in vitro study. However, it has the disadvantages of unstable growth rate and regression time. Therefore, this model can not fully reflect the in vivo human hemangioma and further study needs to be carried out for establishing better model for investigation.